Staphylococcus aureus is a versatile pathogen that causes a wide variety of infections. In addition to its large arsenal of virulence factors, S. aures can grow in biofilm communities that are recalcitrant to antibiotic treatment and host defenses. It is estimated that 80% of S. aureus hospital infections are associated with biofilm. This important aspect of staphylococcal growth and infection has received increased attention, yet we still do not fully understand the molecular mechanisms behind it. Biofilm development can generally be divided into four stages: adherence, accumulation, maturation and dispersion. Mature staphylococcal biofilms are characterized as bacterial communities encased in complex structures comprised of extrapolymeric substances that include extracellular DNA (eDNA) released from active cell death. Biofilm communities are metabolically heterogeneous and the physiological state of each cell is dictated by its location within the different environmental niches (e.g. aerobic, anaerobic). Cells are also actively released from the biofilm, which allows the infection to disseminate to other areas in the host. Previously we have characterized a new gene, msa that is involved in regulation of virulence and biofilm development. In this application, we will test the hypothesis that msa plays a key role in biofilm maturation and/or dispersion by regulating structuring and dispersion factors both in vitro and in vivo. We also hypothesize that the structural defects in biofilm caused by mutation of msa will lead to reduced pathogenic impact in biofilm-associated infections and increased clearance by the host. Finally, since the ultimate goal of this study is to exploit msa as a therapeutic target to enhance conventional therapy, we will examine the impact of msa on biofilm susceptibility to antibiotic treatment in vivo. We have developed two specific aims to test this hypothesis: 1) Define the role of msa in biofilm development in three representative clinical isolates. We will examine the maturation and dispersion stages of biofilm using confocal microscopy and proteomics. We will also map out the expression of msa within biofilm. 2) Define the role of msa in biofilm-associated infections, host responses and susceptibility to antibiotics using a murine model. Findings from this study will lead to development of anti-biofilm therapies that will be critical to combat recalcitrant infections.